Project Summary The goals of this project are to: i) investigate the ecology and evolution of the microbial genus Enterococcus, an evolutionarily ancient lineage of gut bacteria, and ii) provide training and career development opportunities to the Principal Investigator. In the anthropocene era, members of the enterococci have evolved from the gut of antibiotic-treated agricultural animals to emerge as multi-drug resistant (MDR) pathogens in the hospital setting. To understand the emergence of MDR enterococci, the ecological and evolutionary constraints that govern host colonization by Enterococcus spp. must first be understood. Thus, the long-term objectives of this research are to better understand the diversity and host-specificity of Enterococcus in the natural environment, and to understand the evolution of Enterococcus spp. in the context of a novel host environment. The first aim of this project is to develop a strategy to type species of Enterococcus, based on a core and unique gene shared by the species of enterococci sequenced to date. The investigator will then use this novel typing strategy for Enterococcus to profile a collection of 1,000 natural isolates of Enterococcus, to identify novel species diversity. Identified novel species will be used to expand the existing phylogeny of enterococci, which will then be analyzed with comparative genomic approaches to advance current understanding of species diversity and specialization for gut ecologies. The second aim of this proposal is to develop a model for experimental evolution of Enterococcus faecium. This model will leverage the genetic diversity of E. faecium to ask how differences in gene content shape the evolutionary trajectory with which a strain adapts to persistently colonize a novel host. To carry out this aim, a model will be developed to evolve mono-associated strains of Enterococcus faecium in the gut of the wax-worm Galleria mellonella: an invertebrate whose larvae are commonly colonized by species of Enterococcus. The diet of G. mellonella consists of pollen and wax: thus, the model will be informative for studying how the genomic attributes of E. faecium strains promote adaptation in the context of novel, diet-related selection. The genetic and physiological changes of the evolved strains will be compared to their ancestral states using transcriptional (RNA-Seq), and phenotypic (Biolog) analyses, as well as high throughput genotypic (Tn-Seq). Overall, this project will advance the understanding the evolution of Enterococcus in the context of symbiosis, and provide an enriching and worthwhile experience to the Principal Investigator.